N1-methylpseudouridine-incorporated mRNA enhances exogenous protein expression and suppresses immunogenicity in primary human fibroblast-like synoviocytes

Mokuda, Sho; Watanabe, Hirofumi; Kohno, Hiroki; Ishitoku, Michinori; Araki, Kei; Hirata, Shintaro; Sugiyama, Eiji

doi:10.1007/s10616-022-00540-4

Cited by 4 publications

(5 citation statements)

References 41 publications

(52 reference statements)

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“…However, its function in synovial tissues has not been reported. To evaluate FXIII-B function, we overexpressed FXIII-B by introducing IVT mRNA into fibroblasts, using a previously reported method (2023) 43:2 [13]. EGFP IVT mRNA was also used as a control.…”

Section: Elucidation Of Fxiii-b Functions In Fls Using Ivt Mrna Methodsmentioning

confidence: 99%

“…Plasmids coding for FXIII-B (NM_001994.3) or enhanced green fluorescent protein (EGFP) were constructed by inserting full-length cDNA into pcDNA3-A(124) vectors, and in vitro transcribed (IVT) mRNA was prepared as previously described [13]. The insert fragment of the HAtagged F13B open reading frame was purchased from GenScript (Tokyo, Japan).…”

Section: In Vitro Transcribed Mrna Transfection Into Primary Cultured...mentioning

confidence: 99%

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Expression of factor XIII originating from synovial fibroblasts and macrophages induced by interleukin-6 signaling

et al. 2023

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Background Blood coagulation factor XIII (FXIII) promotes cross-linking between fibrin molecules at the final stage of the blood coagulation cascade. However, its expression in cells or tissues and function, particularly factor XIII subunit B (FXIII-B), remains controversial. Hemorrhagic FXIII deficiency following anti-interleukin-6 (IL-6) receptor antibody treatment has been reported in patients with rheumatoid arthritis (RA). Patients receiving this biologics have reduced FXIII activity when compared to the activity in those treated with other biologics. The relationship between pro-inflammatory cytokines and FXIII expression remains unknown. Methods To investigate the expression pattern of FXIII in synovial tissues, immunohistochemistry, RT-qPCR, and western blotting were performed. FXIII-A expressed monocyte-derived macrophages were treated with recombinant IL-6 and anti-IL-6 receptor antibody. RNA sequencing of FXIII-B-overexpressing cells was performed to clarify the function of FXIII-B. Results The immunohistochemical analysis of synovial tissues revealed that factor XIII subunit A (FXIII-A) was expressed in M2 macrophages, and FXIII-B was expressed in fibroblast-like synoviocytes. IL-6 stimulation upregulated FXIII-A expression in IL-4-induced monocyte-derived macrophages, and the anti-IL-6 receptor antibody suppressed FXIII-A expression. FXIII-B was more abundantly secreted in the supernatant of fibroblast-like synoviocytes compared with that of other cells. RNA sequencing showed that FXIII-B elevated the expression of genes associated with anti-apoptotic molecules and chemokines. Conclusions Our findings highlight that synovial tissue is one of the sources of FXIII production. We also have demonstrated IL-6-dependent FXIII-A expression and the novel potential functions of FXIII-B.

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Section: Elucidation Of Fxiii-b Functions In Fls Using Ivt Mrna Methodsmentioning

confidence: 99%

Section: In Vitro Transcribed Mrna Transfection Into Primary Cultured...mentioning

confidence: 99%

Expression of factor XIII originating from synovial fibroblasts and macrophages induced by interleukin-6 signaling

et al. 2023

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“… 23 For example, the incorporation of nucleotide modifications, including 5-methylcytosine (m5C), m6A, 5-methyluridine (m5U), 2-thiouridine (s2U), and N1-methylpseudouridine (m1Ψ), or optimization of free-ending poly(A) and 3’ UTR into linear precursor molecules abrogates the immune response by evading the activation of TLR-3, -7, and -8. 36 , 322 – 324 In addition, substitution with m1Ψ, m6A, and s2U in mRNA molecules suppresses the degradation of RNA by RNase L. 325 Moreover, unmodified mRNA can induce the activation of RNA-dependent protein kinase (PKR). 37 PKR is one of four kinases known to phosphorylate the subunit of translation initiation factor 2 (eIF2) and repress translation.…”

Section: Conclusion and Prospectsmentioning

confidence: 99%

Circular RNA vaccine in disease prevention and treatment

Niu,

Wu,

Lian

2023

Sig Transduct Target Ther

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CircRNAs are a class of single-stranded RNAs with covalently linked head-to-tail topology. In the decades since its initial discovery, their biogenesis, regulation, and function have rapidly disclosed, permitting a better understanding and adoption of them as new tools for medical applications. With the development of biotechnology and molecular medicine, artificial circRNAs have been engineered as a novel class of vaccines for disease treatment and prevention. Unlike the linear mRNA vaccine which applications were limited by its instability, inefficiency, and innate immunogenicity, circRNA vaccine which incorporate internal ribosome entry sites (IRESs) and open reading frame (ORF) provides an improved approach to RNA-based vaccination with safety, stability, simplicity of manufacture, and scalability. However, circRNA vaccines are at an early stage, and their optimization, delivery and applications require further development and evaluation. In this review, we comprehensively describe circRNA vaccine, including their history and superiority. We also summarize and discuss the current methodological research for circRNA vaccine preparation, including their design, synthesis, and purification. Finally, we highlight the delivery options of circRNA vaccine and its potential applications in diseases treatment and prevention. Considering their unique high stability, low immunogenicity, protein/peptide-coding capacity and special closed-loop construction, circRNA vaccine, and circRNA-based therapeutic platforms may have superior application prospects in a broad range of diseases.

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“…The properties of this modification have been widely studied and are currently used to increase the efficiency of therapeutic mRNAs and mRNA vaccines. Earlier, the incorporation of a m1Ψ monomer into mRNA was shown to increase its stability and translation efficiency in multiple mammalian cell lines and mouse models while also reducing cellular innate immune response [20,26,29]. In addition, two COVID-19 mRNA vaccines were developed, in which canonical uridine was replaced with m1Ψ [21].…”

Section: Guide Rnas Modified With M1ψ Can Cleave Plasmid Dna Substratementioning

confidence: 99%

“…In addition, m1Ψ is widely used to design mRNA vaccines against other pathogens, such as HIV-1, Zika, and Ebola [23,24]. The incorporation of m1Ψ into mRNA provides a several-fold increase in protein expression in cell cultures or mice while simultaneously reducing cytotoxicity and innate immune response in vivo [25,26]. This modification contributes to many biological processes: RNA stability, translation, RNA-protein interactions, and innate immunity [20,27].…”

Section: Guide Rnas Modified With M1ψ Can Cleave Plasmid Dna Substratementioning

confidence: 99%

Influence of N1-Methylpseudouridine in Guide RNAs on CRISPR/Cas9 Activity

Prokhorova,

Matveeva,

Zakabunin

et al. 2023

IJMS

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At present, there are many strategies to improve the activity of CRISPR/Cas9. A well-known and effective approach is guide RNA modification. Many chemical guide RNA modifications have been studied, whereas naturally occurring RNA modifications are largely unexplored. N1-methylpseudouridine (m1Ψ) is an RNA base modification widely used in mRNA therapy, and it holds great promise for application in genome editing systems. The present study focuses on investigating the effect of N1-methylpseudouridine on the functioning of CRISPR/Cas9. In vitro cleavage assays helped determine the level of m1Ψ guide RNA modification that is sufficient to cleave the target substrate. By analyzing FAM-labeled dsDNA substrate cleavage, we calculated the kinetic parameters and the specificity scores of modified guide RNAs. Neon transfection and digital PCR enabled us to assess the activity of modified guide RNAs in mammalian cells. Our study shows that the presence of m1Ψ in guide RNAs can help preserve on-target genome editing while significantly reducing the off-target effects of CRISPR/Cas9 in vitro. We also demonstrate that Cas9 complexes with guide RNAs containing m1Ψ allow for genome editing in human cells. Thus, the incorporation of m1Ψ into guide RNAs supports CRISPR/Cas9 activity both in vitro and in cells.

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N1-methylpseudouridine-incorporated mRNA enhances exogenous protein expression and suppresses immunogenicity in primary human fibroblast-like synoviocytes

Cited by 4 publications

References 41 publications

Expression of factor XIII originating from synovial fibroblasts and macrophages induced by interleukin-6 signaling

Expression of factor XIII originating from synovial fibroblasts and macrophages induced by interleukin-6 signaling

Circular RNA vaccine in disease prevention and treatment

Influence of N1-Methylpseudouridine in Guide RNAs on CRISPR/Cas9 Activity

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